1. Field of the Invention
The present invention relates to an L-glutamine-producing bacterium belonging to coryneform bacteria, and which is useful for production of L-glutamine. The present invention also relates to a method for producing L-glutamine. L-glutamine is an industrially useful amino acid as an ingredient in seasonings, liver function promoting agents, amino acid infusions, comprehensive amino acid pharmaceuticals and so forth.
2. Brief Description of the Related Art
Various techniques for increasing L-amino acid production using recombinant DNA techniques have been disclosed. For example, techniques for enhancing activities of enzymes involved in L-amino acid biosyntheses (WO96/40934), or techniques for reducing activities of enzymes involved in degradation of L-amino acids (WO/96/17930), and so fort, are known. A method for producing L-glutamine using a coryneform bacterium having enhanced glutamine synthetase activity has been disclosed (US Pat App Pub No. 2003/0003550). Furthermore, genes encoding glutamine synthetase (Genbank Accession No. Y13221) and glutamine 2-oxoglutarate aminotranferase (Genbank Accession No. AB024708) have been reported (FEMS Microbiol. Le (1997) 154(1) 81-88, Microbiology (2001) 147, 2961-2970), and are known to be involved in glutamine biosynthesis and degradation of coryneform bacteria.
Besides the aforementioned genes, existence of an enzyme involved in degradation of L-glutamine in coryneform bacteria has been suggested (Amino Acids, 7:73-77 (1963)). However, this enzyme is inhibited by ammonium ions and low pH, and thus barely functions in glutamine fermentation in the presence of the required ammonium ions.
Glutamine (glutamine amidohydrolase) is known as an enzyme that degrades L-glutamine by hydrolysis. Genes encoding glutaminase have been reported for Pseudomonas bacteria (FEMS Microbiol. lett., 178 (2), 327-335 (1999)), Aspergillus oryzae (Appl. Microbiol. Biotechnol., 54, 59-68 (2000), EP 1 077 256 A1), Rhizobium etli (Biochim. Biophys. Acta, 1444 (3): 451-6, 1999), rat (J. Biol. Chem., 266(28), 18792-18796(1991)), amongothers. Furthermore, existence of glutaminase activity in Escherichia coli has been reported (J. Biol. Chem., 243 (5) 853-878 (1968)). However, a glutaminase gene derived from coryneform bacteria has not been identified, and any effect mutation of such a gene will have on glutamine production is not known.
A method for enhancing gene expression by modifying a promoter sequence of the desired gene is known (Japanese Patent Laid-open (Kokai) No. 2000-818935). Furthermore, a gene encoding glutamine synthetase (hereiner, “glnA”) from coryneform bacteria is known (FEMS Microbiology Letters, 154, 81-88, 1997). Moreover, the transcription initiation site of the gene including the promoter region has been identified (FEMS Microbiology Letters, 205, 361-367, 2001). However, enhancing the expression of the glutamine synthetase gene by modifying a promoter sequence has not been previously described.
Methods of improving microorganisms by breeding have frequently been used in fermentation production of L-amino acids. That is, since the yield of L-amino acids produced from wild-type microorganisms is often extremely low, methods of imparting auxotrophic or analogue resistance by mutation, or imparting mutations designed to improve metabolic regulation, or a combination of these are known. Although L-glutamine can be obtained by these known methods, there is clearly a need in the art to improve fermentation yields so that production of L-glutamine can be accomplished efficiently and at a low cost.